HPLC: Isocratic or Gradient Elution and Assessment of Linearity In Analytical Methods

The aim of pharmaceutical analysis is to obtain the necessary qualitative and quantitative information about the investigated sample. HPLC is the dominant separation technique in modern pharmaceutical and biomedical analysis because it results in highly efficient separations and in most cases provides high detection sensitivity. Chromatographic optimization procedures are becoming more multidisciplinary to obtain more and more information on the separations which may be isocratic or gradient. During validation of analytical methods a correlation coefficient close to unity (r = 1) is considered sufficient evidence to conclude that the experimenter has a perfect linear calibration since it is common practice to check the linearity of a calibration curve by inspection of the correlation coefficient. The aim of the presented review is divided into two parts, first is to compare isocratic and gradient elution and to propose suitable statistical procedures to access linearity of the analytical method. We found that many of the previous reasons for avoiding gradient elution (i.e. long re-equilibration times, poor precision and difficult optimization) appear much too pessimistic and linear functional analysis or lack of fitness test as another suitable statistical tool to evaluate linearity of the analytical method. Some other validation parameters are also discussed here in brief.